HIV encephalitis and AIDS dementia complex are neurological disorders that continue to afflict AIDS patients even in the presence of highly active retroviral therapies. Research has suggested that ethanol may exacerbate these symptoms and may do so by modulating the susceptibility of the blood-brain barrier (BBB) to infiltration by HIV infected monocytes or free HIV virus. In order to study these complex interactions, an in vitro model that accurately represents the in vivo BBB is required. When brain microvascular endothelial cell are cultured in vitro, however, they undergo a great deal of de-differentiation and lose many of the specialized biochemical and morphological features observed in vivo. The overall goal of the proposed research is to gain a sophisticated genome-wide understanding of the determinants of the in vivo BBB phenotype that are lost in vitro and progress towards truly representative in vitro BBB models by recreating the in vivo environment. Functional genomics in the form of suppression subtractive hybridization will be used to elucidate the differential gene expression profiles between intact brain microvessels (BBB) and primary cultures of brain microvessel endothelial cells. This will allow the identification of functional clusters of known and novel genes that elicit the unique in vivo BBB phenotype. Armed with this global functional profile, a gene microarray will be created and used as a diagnostic for the quantitative assessment of an in vitro model's ability to reestablish in vivo conditions. Perivascular brain cells can restore some BBB properties when co-cultured with brain microvascular endothelial cells in vitro. The contributions of astrocytes, neurons, and pericytes on the in vitro phenotype will be assessed using the diagnostic gene microarray. Molecular pathways and physiological characteristics that are reestablished by co-culture will be identified and provide targets for rational tuning of in vitro models. Finally, the effects of ethanol on the functional features of the in vitro model will be investigated as a preliminary validation of in vitro models for the study of the complicated interactions between HIV, ethanol, and the BBB.